Natriuretic polypeptides for reducing or preventing restenosis

ABSTRACT

Materials and methods related to using natriuretic polypeptides to reduce proliferation of smooth muscle cells, and to reduce or prevent restenosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 61/141,127, filed on Dec. 29, 2008.

TECHNICAL FIELD

This document relates to natriuretic polypeptides. For example, thisdocument provides methods and materials related to natriureticpolypeptides and the use of natriuretic polypeptides to preventrestenosis.

BACKGROUND

Natriuretic polypeptides (NPs) are polypeptides that can causenatriuresis (increased sodium excretion in the urine). Such polypeptidescan be produced by brain, heart, kidney, and/or vascular tissue. Thenatriuretic peptide family in humans includes the cardiac hormonesatrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP),C-type natriuretic peptide (CNP), and urodilatin (URO). Natriureticpolypeptides function via well-characterized guanylyl cyclase receptors(i.e., NPR-A for ANP, BNP, and URO; and NPR-B for CNP) and the secondmessenger cyclic 3′5′ guanosine monophosphate (cGMP) (Kuhn (2003) CircRes 93:700-709; Tawaragi et al. (1991) Biochem. Biophys. Res. Commun.175:645-651; and Komatsu et al. (1991) Endocrinol. 129:1104-1106).

SUMMARY

This document is based in part on the discovery that both naturallyoccurring and synthetic natriuretic polypeptides can haveantiproliferative actions on human aortic vascular smooth muscle cells(HAoSMC) in culture. Thus, natriuretic polypeptides may be useful toreduce or prevent restenosis, for example.

In one aspect, this document features a method for reducing restenosisin a subject identified as being in need thereof, comprisingadministering to the subject a composition comprising arestenosis-reducing amount of a composition comprising apharmaceutically acceptable carrier and a natriuretic polypeptide. Thenatriuretic polypeptide can be a chimeric natriuretic polypeptidecomprising (a) the ring structure of a first natriuretic polypeptide ora variant of the ring structure of the first natriuretic polypeptide,and (b) an amino acid sequence from a second natriuretic polypeptide ora variant of the amino acid sequence from the second natriureticpolypeptide. The natriuretic polypeptide can comprise the amino acidsequence set forth in SEQ ID NO:1, but with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:1, the amino acid sequence set forth in SEQ ID NO:2, but with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:2, the amino acid sequence set forth inSEQ ID NO:3, but with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:3, theamino acid sequence set forth in SEQ ID NO:9, or the amino acid sequenceset forth in SEQ ID NO:9 with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:9, theamino acid sequence set forth in SEQ ID NO:13, or the amino acidsequence set forth in SEQ ID NO:13 with one, two, three, four, or fiveamino acid substitutions relative to the sequence set forth in SEQ IDNO:13, the amino acid sequence set forth in SEQ ID NO:27, or the aminoacid sequence set forth in SEQ ID NO:27 with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:27, the amino acid sequence set forth in SEQ ID NO:29, or theamino acid sequence set forth in SEQ ID NO:29 with one, two, three,four, or five amino acid substitutions relative to the sequence setforth in SEQ ID NO:29, or the amino acid sequence set forth in SEQ IDNO:30, or the amino acid sequence set forth in SEQ ID NO:30 with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:30.

The method can include administering the composition as a continuousintravenous infusion (e.g., for about one to about seven days). Themethod can include administering the composition as a continuousintravenous infusion for about one to about seven days, and subsequentlyadministering the composition subcutaneously for about five to about 30days. The method can include administering the composition as acontinuous intravenous infusion at a dose of about 0.1 ng polypeptide/kgbody mass/minute to about 30 ng polypeptide/kg body mass/minute, andsubsequently administering the composition subcutaneously at a dose ofabout 10 ng polypeptide/kg body mass/day to about 30 ng polypeptide/kgbody mass/day. The method can include administering the composition as acontinuous intravenous infusion at a dose of about 0.1 ng polypeptide/kgbody mass/minute to about 30 ng polypeptide/kg body mass/minute forabout three hours to about seven days, and subsequently administeringthe composition subcutaneously at a dose of about 10 ng polypeptide/kgbody mass/day to about 30 ng polypeptide/kg body mass/day for about fiveto about 30 days. The subject can be an angioplasty or stent placementpatient. The method can include administering the continuous intravenousinfusion beginning at or about the time of reperfusion. The compositioncan be administered beginning about three hours after the onset ofreperfusion. The composition can be administered from about three hoursto about 12 hours after reperfusion. The method can includeadministering the composition at a dose of about 1 ng polypeptide/kgbody mass/minute to about 30 ng polypeptide/kg body mass/minute. Thecomposition can be on an implanted medical device (e.g., a stent).

In another aspect, this document features a method for reducingproliferation of smooth muscle cells, comprising contacting the smoothmuscle cells with an effective amount of a composition comprising apharmaceutically acceptable carrier and a natriuretic polypeptide. Thenatriuretic polypeptide can be a chimeric natriuretic polypeptidecomprising (a) the ring structure of a first natriuretic polypeptide ora variant of the ring structure of the first natriuretic polypeptide,and (b) an amino acid sequence from a second natriuretic polypeptide ora variant of the amino acid sequence from said second natriureticpolypeptide. The natriuretic polypeptide can comprise the amino acidsequence set forth in SEQ ID NO:1, but with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:1, the amino acid sequence set forth in SEQ ID NO:2, but with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:2, the amino acid sequence set forth inSEQ ID NO:3, but with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:3, theamino acid sequence set forth in SEQ ID NO:9, or the amino acid sequenceset forth in SEQ ID NO:9 with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:9, theamino acid sequence set forth in SEQ ID NO:13, or the amino acidsequence set forth in SEQ ID NO:13 with one, two, three, four, or fiveamino acid substitutions relative to the sequence set forth in SEQ IDNO:13, the amino acid sequence set forth in SEQ ID NO:27, or the aminoacid sequence set forth in SEQ ID NO:27 with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:27, the amino acid sequence set forth in SEQ ID NO:29, or theamino acid sequence set forth in SEQ ID NO:29 with one, two, three,four, or five amino acid substitutions relative to the sequence setforth in SEQ ID NO:29, or the amino acid sequence set forth in SEQ IDNO:30, or the amino acid sequence set forth in SEQ ID NO:30 with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:30.

This document also features the use of a natriuretic polypeptide and apharmaceutically acceptable carrier in the manufacture of a medicamentfor reducing restenosis. The natriuretic polypeptide can be a chimericnatriuretic polypeptide comprising (a) the ring structure of a firstnatriuretic polypeptide or a variant of the ring structure of said firstnatriuretic polypeptide, and (b) an amino acid sequence from a secondnatriuretic polypeptide or a variant of said amino acid sequence fromsaid second natriuretic polypeptide. The natriuretic polypeptide cancomprise the amino acid sequence set forth in SEQ ID NO:1, but with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:1, the amino acid sequence set forth inSEQ ID NO:2, but with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:2, theamino acid sequence set forth in SEQ ID NO:3, but with one, two, three,four, or five amino acid substitutions relative to the sequence setforth in SEQ ID NO:3, the amino acid sequence set forth in SEQ ID NO:9,or the amino acid sequence set forth in SEQ ID NO:9 with one, two,three, four, or five amino acid substitutions relative to the sequenceset forth in SEQ ID NO:9, the amino acid sequence set forth in SEQ IDNO:13, or the amino acid sequence set forth in SEQ ID NO:13 with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:13, the amino acid sequence set forth inSEQ ID NO:27, or the amino acid sequence set forth in SEQ ID NO:27 withone, two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:27, the amino acid sequence set forth inSEQ ID NO:29, or the amino acid sequence set forth in SEQ ID NO:29 withone, two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:29, or the amino acid sequence set forthin SEQ ID NO:30, or the amino acid sequence set forth in SEQ ID NO:30with one, two, three, four, or five amino acid substitutions relative tothe sequence set forth in SEQ ID NO:30.

The medicament can be formulated for intravenous infusion (e.g.,continuous intravenous infusion for about one to about seven days). Themedicament can be formulated for continuous intravenous infusion forabout one to about seven days, and subsequent subcutaneousadministration for about five to about 30 days (e.g., continuousintravenous infusion at a dose of about 0.1 ng polypeptide/kg bodymass/minute to about 30 ng polypeptide/kg body mass/minute, andsubsequent subcutaneous administration at a dose of about 10 ngpolypeptide/kg body mass/day to about 30 ng polypeptide/kg bodymass/day, or continuous intravenous infusion at a dose of about 0.1 ngpolypeptide/kg body mass/minute to about 30 ng polypeptide/kg bodymass/minute for about three hours to about seven days, and subsequentsubcutaneous administration at a dose of about 10 ng polypeptide/kg bodymass/day to about 30 ng polypeptide/kg body mass/day for about five toabout 30 days). The medicament can be on an implantable medical device(e.g., a stent).

In yet another aspect, this document features the use of a natriureticpolypeptide and a pharmaceutically acceptable carrier in the manufactureof a medicament for reducing proliferation of smooth muscle cells. Thenatriuretic polypeptide can be a chimeric natriuretic polypeptidecomprising (a) the ring structure of a first natriuretic polypeptide ora variant of the ring structure of said first natriuretic polypeptide,and (b) an amino acid sequence from a second natriuretic polypeptide ora variant of said amino acid sequence from said second natriureticpolypeptide. The natriuretic polypeptide can comprise the amino acidsequence set forth in SEQ ID NO:1, but with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:1, the amino acid sequence set forth in SEQ ID NO:2, but with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:2, the amino acid sequence set forth inSEQ ID NO:3, but with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:3, theamino acid sequence set forth in SEQ ID NO:9, or the amino acid sequenceset forth in SEQ ID NO:9 with one, two, three, four, or five amino acidsubstitutions relative to the sequence set forth in SEQ ID NO:9, theamino acid sequence set forth in SEQ ID NO:13, or the amino acidsequence set forth in SEQ ID NO:13 with one, two, three, four, or fiveamino acid substitutions relative to the sequence set forth in SEQ IDNO:13, the amino acid sequence set forth in SEQ ID NO:27, or the aminoacid sequence set forth in SEQ ID NO:27 with one, two, three, four, orfive amino acid substitutions relative to the sequence set forth in SEQID NO:27, the amino acid sequence set forth in SEQ ID NO:29, or theamino acid sequence set forth in SEQ ID NO:29 with one, two, three,four, or five amino acid substitutions relative to the sequence setforth in SEQ ID NO:29, or the amino acid sequence set forth in SEQ IDNO:30, or the amino acid sequence set forth in SEQ ID NO:30 with one,two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:30.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing showing the amino acid sequences and structures ofANP (SEQ ID NO:1), BNP (SEQ ID NO:2), CNP (SEQ ID NO:3), and DNP (SEQ IDNO:5).

FIG. 2 is a graph plotting the effect of natriuretic polypeptides onHAoSMC proliferation.

DETAILED DESCRIPTION Natriuretic Compounds

This document provides natriuretic compounds (e.g., polypeptides) andcompositions that can be used to increase natriuretic activity in asubject in need thereof. For example, isolated NPs can increase plasmacGMP levels, urinary cGMP excretion, net renal cGMP generation, urineflow, urinary sodium excretion, urinary potassium excretion, hematocrit,plasma BNP immunoreactivity, renal blood flow, and/or plasma ANPimmunoreactivity, and decrease renal vascular resistance, proximal anddistal fractional reabsorption of sodium, mean arterial pressure,pulmonary capillary wedge pressure, right atrial pressure, pulmonaryarterial pressure, plasma renin activity, plasma angiotensin II levels,plasma aldosterone levels, renal perfusion pressure, and/or systemicvascular resistance. As described herein, NPs also may be useful toreduce or prevent restenosis that can occur, for example, after vascularsurgery, cardiac surgery, interventional radiology, or interventionalcardiology following angioplasty or stent placement. For example, ANP,BNP, CNP, and the designer natriuretic polypeptides CD-NP, CU-NP,ABC-NP, ABC-NP1, and BC-NP2 described herein can reduce proliferation ofHAoSMC, as described in the Example below.

As used herein, the term “natriuretic polypeptide” or “NP” includesnative (naturally occurring, wild type) NPs (e.g., ANP, BNP, CNP, andURO, as well as Dendroaspis natriuretic peptide (DNP)), one or moreportions of a native NP, variants of a native NP, or chimeras of nativeNPs, portions of native NPs, or variants of native NPs or portions ofnative NPs. In some embodiments, a NP includes only portions of themature form of a native NP. Chimeric NPs containing amino acid sequencesfrom human CNP, BNP, and ANP or URO, or Dendroaspis DNP, can beparticularly useful, although other NPs are contemplated herein.

The term “isolated polypeptide” refers to a polypeptide that (1) is notassociated with proteins found in nature, (2) is free of other proteinsfrom the same source (e.g., free of human proteins), (3) is expressed bya cell from a different species, or (4) does not occur in nature. Anisolated polypeptide can be, for example, encoded by DNA or RNA,including synthetic DNA or RNA, or some combination thereof.

Amino acid sequences for endogenous human mature NPs include thefollowing:

(SEQ ID NO: 1) ANP: SLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 2) BNP:SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 3) CNP:GLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 4) URO:TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY

In addition, the native Dendroaspis amino acid sequence for DNP is

(SEQ ID NO: 5) EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA

Chimeric NPs can include amino acid sequences from two or moreindividual NPs. In some embodiments, for example, a chimeric polypeptidecan include amino acid sequences from ANP and CNP; BNP and CNP; ANP,BNP, and CNP; CNP and URO; CNP and DNP; or CNP, URO, and BNP. In somecases, a chimeric NP can include a ring structure and cysteine bond(e.g., the ring structure and cysteine bond of ANP, BNP, CNP, or DNP) incombination with one or more amino acid segments from another NP. Thechimeric NPs described herein are non-limiting examples of polypeptidesthat can be useful to prevent or reduce restenosis, for example.

In some embodiments, a chimeric BD-NP can include the N-terminal 26amino acids of human BNP (SPKMVQGSGCFGRKMDRISSSSGLGC; SEQ ID NO:6) andthe C-terminal 15 amino acids of DNP (PSLRDPRPNAPSTSA; SEQ ID NO:7), andcan have the amino acid sequenceSPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:8).

In some embodiments, a chimeric CD-NP can include the amino acidsequence of human CNP (GLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO:3) and theC-terminal 15 amino acids of DNP (PSLRDPRPNAPSTSA; SEQ ID NO:7), and canhave the amino acid sequence GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQID NO:9).

In some embodiments, a chimeric CU-NP can include the N-terminal tenamino acids of human URO (TAPRSLRRSS; SEQ ID NO:10), the 17 amino acidring structure and disulfide bond of human CNP (CFGLKLDRIGSMSGLGC; SEQID NO:11), and the C-terminal five amino acids of human URO (NSFRY; SEQID NO:12), and can have the amino acid sequenceTAPRSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO:13).

In some embodiments, a chimeric BAA-NP can include the N-terminal sixamino acids of human ANP (SLRRSS; SEQ ID NO:14), the 17 amino acid ringstructure and disulfide bond of human BNP (CFGRKMDRISSSSGLGC; SEQ IDNO:15), and the C-terminal five amino acids of human ANP (NSFRY; SEQ IDNO:12), and can have the amino acid sequenceSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO:16).

In some embodiments, a chimeric BUA-NP can include the N-terminal 10amino acids of human URO (TAPRSLRRSS; SEQ ID NO:10), the 17 amino acidring structure and disulfide bond of human BNP (CFGRKMDRISSSSGLGC; SEQID NO:15), and the C-terminal 5 amino acids of human ANP (NSFRY; SEQ IDNO:12), and can have the amino acid sequenceTAPRSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO:17).

In some embodiments, a chimeric CAA-NP can include the N-terminal 6amino acids of human ANP (SLRRSS; SEQ ID NO:14), the 17 amino acid ringstructure and disulfide bond of human CNP (CFGLKLDRIGSMSGLGC; SEQ IDNO:11), and the C-terminal 5 amino acids of human ANP (NSFRY; SEQ IDNO:12), and can have the amino acid sequenceSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO:18).

As another example, in some embodiments, a chimeric CAB-NP can includethe N-terminal six amino acids of human ANP (SLRRSS; SEQ ID NO:14), the17 amino acid ring structure and disulfide bond of human CNP(CFGLKLDRIGSMSGLGC; SEQ ID NO:11), and the C-terminal six amino acids ofhuman BNP (KVLRRH; SEQ ID NO:19), and can have the amino acid sequenceSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO:20).

In some embodiments, a chimeric CBB-NP can include the N-terminal nineamino acids of human BNP (SPKMVQGSG; SEQ ID NO:21), the 17 amino acidring structure and disulfide bond of human CNP (CFGLKLDRIGSMSGLGC; SEQID NO:11), and the C-terminal six amino acids of human BNP (KVLRRH; SEQID NO:19), and can have the amino acid sequenceSPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO:22).

In some embodiments, a chimeric CDD-NP can include the N-terminal sixamino acids of DNP (EVKYDP; SEQ ID NO:23), the 17 amino acid ringstructure and disulfide bond of human CNP (CFGLKLDRIGSMSGLGC; SEQ IDNO:11), and the C-terminal 15 amino acids of DNP (PSLRDPRPNAPSTSA; SEQID NO:7), and can have the amino acid sequenceEVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:24).

In some embodiments, a chimeric CUB-NP can include the N-terminal 10amino acids of human URO (TAPRSLRRSS; SEQ ID NO:10), the 17 amino acidring structure and disulfide bond of human CNP (CFGLKLDRIGSMSGLGC; SEQID NO:11), and the C-terminal six amino acids of human BNP (KVLRRH; SEQID NO:19), and can have the amino acid sequenceTAPRSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO:25).

In some embodiments, a chimeric ABC-NP1 can include amino acids 11 to 15of human ANP (RMDRI; SEQ ID NO:26) at its amino terminus, followed bythe amino acid sequence of human CNP (GLSKGCFGLKLDRIGSMSGLGC; SEQ IDNO:3), and the C-terminal six amino acids of human BNP (KVLRRH; SEQ IDNO:19), and can have the amino acid sequenceRMDRIGLSKGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO:27).

In some cases, a chimeric NP can include a variant (e.g., asubstitution, addition, or deletion) at one or more positions (e.g.,one, two, three, four, five, six, seven, eight, nine, or ten positions)with respect to any of SEQ ID NOS:1 to 27. For example, a chimericABC-NP can include amino acids 11 to 15 of human ANP (RMDRI; SEQ IDNO:26) at its amino terminus, followed by the amino acid sequence ofhuman CNP with the exception that the amino acid residues at positions15, 16, and 17 are changed to Arg, Glu, and Ala (GLSKGCFGLKLDRIREASGLGC;SEQ ID NO:28), and the C-terminal six amino acids of human BNP (KVLRRH;SEQ ID NO:19), and can have the amino acidRMDRIGLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO:29). A chimeric BC-NP2 caninclude the amino acid sequence of human CNP with the exception that theamino acid residues at positions 15, 16, and 17 are changed to Arg, Glu,and Ala (GLSKGCFGLKLDRIREASGLGC; SEQ ID NO:28), followed by theC-terminal six amino acids of human BNP (KVLRRH; SEQ ID NO:19), and canhave the amino acid sequence GLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ IDNO:30).

Variant NPs, e.g., those having one or more amino acid substitutionsrelative to a native NP amino acid sequence, can be prepared andmodified as described herein. Amino acid substitutions can be made, insome cases, by selecting substitutions that do not differ significantlyin their effect on maintaining (a) the structure of the peptide backbonein the area of the substitution, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Forexample, naturally occurring residues can be divided into groups basedon side-chain properties: (1) hydrophobic amino acids (norleucine,methionine, alanine, valine, leucine, and isoleucine); (2) neutralhydrophilic amino acids (cysteine, serine, and threonine); (3) acidicamino acids (aspartic acid and glutamic acid); (4) basic amino acids(asparagine, glutamine, histidine, lysine, and arginine); (5) aminoacids that influence chain orientation (glycine and proline); and (6)aromatic amino acids (tryptophan, tyrosine, and phenylalanine)Substitutions made within these groups can be considered conservativesubstitutions. Non-limiting examples of useful substitutions include,without limitation, substitution of valine for alanine, lysine forarginine, glutamine for asparagine, glutamic acid for aspartic acid,serine for cysteine, asparagine for glutamine, aspartic acid forglutamic acid, proline for glycine, arginine for histidine, leucine forisoleucine, isoleucine for leucine, arginine for lysine, leucine formethionine, leucine for phenyalanine, glycine for proline, threonine forserine, serine for threonine, tyrosine for tryptophan, phenylalanine fortyrosine, and/or leucine for valine.

Non-limiting examples of variant chimeric NPs include the following:

(SEQ ID NO: 31) TLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 32)SIRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 33)SLKRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 34)SLRKSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 35)SLRRSSCFGGRMDRIGAQSGLGCNTFRY (SEQ ID NO: 36)SLRRSSCFGGRMDRIGAQSGLGCNSLRY (SEQ ID NO: 37)SLRRSSCFGGRMDRIGAQSGLGCNSFKY (SEQ ID NO: 38)SLRRSSCFGGRMDRIGAQSGLGCNSFRF (SEQ ID NO: 39)TPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 40)SGKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 41)SPRMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 42)SPKLVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 43)SPKMVQGSGCFGRKMDRISSSSGLGCKVIRRH (SEQ ID NO: 44)SPKMVQGSGCFGRKMDRISSSSGLGCKVLKRH (SEQ ID NO: 45)SPKMVQGSGCFGRKMDRISSSSGLGCKVLRKH (SEQ ID NO: 46)SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRR (SEQ ID NO: 47) PLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 48) GISKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49)GLTKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 50) GLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 51) GLSKGCFGLKLDRIGSMSPLGC (SEQ ID NO: 52)GLSKGCFGLKLDRIGSMSGIGC (SEQ ID NO: 53) GLSKGCFGLKLDRIGSMSGLPC(SEQ ID NO: 54) GLSKGCFGLKLDRIGSMSGLGS (SEQ ID NO: 55)TPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 56)SGKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 57)SPRMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 58)SPKLVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 59)SPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPTTSA (SEQ ID NO: 60)SPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSSSA (SEQ ID NO: 61)SPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTTA (SEQ ID NO: 62)SPKMVQGSGCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSV (SEQ ID NO: 63)PLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 64)GISKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 65)GLTKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 66)GLSRGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 67)GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPTTSA (SEQ ID NO: 68)GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSSSA (SEQ ID NO: 69)GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTTA (SEQ ID NO: 70)GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSV (SEQ ID NO: 71)SAPRSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO: 72)TVPRSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO: 73)TAGRSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO: 74)TAPKSLRRSSCFGLKLDRIGSMSGLGCNSFRY (SEQ ID NO: 75)TAPRSLRRSSCFGLKLDRIGSMSGLGCNTFRY (SEQ ID NO: 76)TAPRSLRRSSCFGLKLDRIGSMSGLGCNSLRY (SEQ ID NO: 77)TAPRSLRRSSCFGLKLDRIGSMSGLGCNSFKY (SEQ ID NO: 78)TAPRSLRRSSCFGLKLDRIGSMSGLGCNSFRF (SEQ ID NO: 79)TLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 80)SIRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 81)SLKRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 82)SLRKSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 83)SLRRSSCFGRKMDRISSSSGLGCNTFRY (SEQ ID NO: 84)SLRRSSCFGRKMDRISSSSGLGCNSLRY (SEQ ID NO: 85)SLRRSSCFGRKMDRISSSSGLGCNSFKY (SEQ ID NO: 86)SLRRSSCFGRKMDRISSSSGLGCNSFRF (SEQ ID NO: 87)SAPRSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 88)TVPRSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 89)TAGRSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 90)TAPKSLRRSSCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 91)TAPRSLRRSSCFGRKMDRISSSSGLGCNTFRY (SEQ ID NO: 92)TAPRSLRRSSCFGRKMDRISSSSGLGCNSLRY (SEQ ID NO: 93)TAPRSLRRSSCFGRKMDRISSSSGLGCNSFKY (SEQ ID NO: 94)TAPRSLRRSSCFGRKMDRISSSSGLGCNSFRF (SEQ ID NO: 95)TLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 96)SIRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 97)SLKRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 98)SLRKSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 99)SLRRSSCFGLKLDRIGSMSGLGCKVIRRH (SEQ ID NO: 100)SLRRSSCFGLKLDRIGSMSGLGCKVLKRH (SEQ ID NO: 101)SLRRSSCFGLKLDRIGSMSGLGCKVLRKH (SEQ ID NO: 102)SLRRSSCFGLKLDRIGSMSGLGCKVLRRR (SEQ ID NO: 103)TPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 104)SGKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 105)SPRMVQGSGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 106)SPKLVQGSGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 107)SPKMVQGSGCFGLKLDRIGSMSGLGCKVIRRH (SEQ ID NO: 108)SPKMVQGSGCFGLKLDRIGSMSGLGCKVLKRH (SEQ ID NO: 109)SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRKH (SEQ ID NO: 110)SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRR (SEQ ID NO: 111)DVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 112)ELKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 113)EVRYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 114)EVKFDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 115)EVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPTTSA (SEQ ID NO: 116)EVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSSSA (SEQ ID NO: 117)EVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTTA (SEQ ID NO: 118)EVKYDPCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSV (SEQ ID NO: 119)SAPRSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 120)TVPRSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 121)TAGRSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 122)TAPKSLRRSSCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 123)TAPRSLRRSSCFGLKLDRIGSMSGLGCKVIRRH (SEQ ID NO: 124)TAPRSLRRSSCFGLKLDRIGSMSGLGCKVLKRH (SEQ ID NO: 125)TAPRSLRRSSCFGLKLDRIGSMSGLGCKVLRKH (SEQ ID NO: 126)TAPRSLRRSSCFGLKLDRIGSMSGLGCKVLRRR (SEQ ID NO: 127)KMDRIGLSKGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 128)RLDRIGLSKGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 129)RMERIGLSKGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 130)RMDKIGLSKGCFGLKLDRIGSMSGLGCKVLRRH (SEQ ID NO: 131)RMDRIGLSKGCFGLKLDRIGSMSGLGCKVIRRH (SEQ ID NO: 132)RMDRIGLSKGCFGLKLDRIGSMSGLGCKVLKRH (SEQ ID NO: 133)RMDRIGLSKGCFGLKLDRIGSMSGLGCKVLRKH (SEQ ID NO: 134)RMDRIGLSKGCFGLKLDRIGSMSGLGCKVLRRR (SEQ ID NO: 135)KMDRIGLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 136)RLDRIGLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 137)RMERIGLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 138)RMDKIGLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 139)RMDRIGLSKGCFGLKLDRIREASGLGCKVIRRH (SEQ ID NO: 140)RMDRIGLSKGCFGLKLDRIREASGLGCKVLKRH (SEQ ID NO: 141)RMDRIGLSKGCFGLKLDRIREASGLGCKVLRKH (SEQ ID NO: 142)RMDRIGLSKGCFGLKLDRIREASGLGCKVLRRR (SEQ ID NO: 143)PLSKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 144)GISKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 145)GLTKGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 146)GLSRGCFGLKLDRIREASGLGCKVLRRH (SEQ ID NO: 147)GLSKGCFGLKLDRIREASGLGCKVIRRH (SEQ ID NO: 148)GLSKGCFGLKLDRIREASGLGCKVLKRH (SEQ ID NO: 149)GLSKGCFGLKLDRIREASGLGCKVLRKH (SEQ ID NO: 150)GLSKGCFGLKLDRIREASGLGCKVLRRR

Further examples of conservative substitutions that can be made at anyposition within the polypeptides provided herein are set forth in Table1.

TABLE 1 Examples of conservative amino acid substitutions OriginalPreferred Residue Exemplary substitutions substitutions Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu CysSer Ser Gln Asn Asn Glu Asp Asp Gly Pro Pro His Asn, Gln, Lys, Arg ArgIle Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val,Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu,Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleucine Leu

In some embodiments, a NP can include one or more non-conservativesubstitutions. Non-conservative substitutions typically entailexchanging a member of one of the classes described above for a memberof another class. Such production can be desirable to provide largequantities or alternative embodiments of such compounds. Whether anamino acid change results in a functional polypeptide can readily bedetermined by assaying the specific activity of the peptide variant.

A polypeptide provided herein can have any sequence and can have anylength. For example, a polypeptide can include the sequences set forthin SEQ ID NOS:6 and 7. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:6 with five or less (e.g., five or less, four or less,three or less, two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof, and (b) the sequenceset forth in SEQ ID NO:7 with three or less (e.g., three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:8, with the exception thatthe first serine residue or the last alanine residue of SEQ ID NO:8 isdeleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:3 and 7. In some cases, a polypeptide provided herein can containan amino acid sequence that aligns to (a) the sequence set forth in SEQID NO:3 with five or less (e.g., five or less, four or less, three orless, two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof, and (b) the sequence set forthin SEQ ID NO:7 with three or less (e.g., three or less, two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:9, with the exception thatthe first glycine residue or the last alanine residue of SEQ ID NO:9 isdeleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:10, 11, and 12. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:10 with three or less (e.g., three or less, two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, (b) the sequence set forth in SEQ ID NO:11 withfive or less (e.g., five or less, four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, and (c) the sequence set forth in SEQ ID NO:12with two or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:13, with the exception that the first threonine residue or the lasttyrosine residue of SEQ ID NO:13 is deleted or replaced with a differentamino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:14, 15, and 12. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:14 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:15 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:12 with two or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:16, with the exception thatthe first serine residue or the last tyrosine residue of SEQ ID NO:16 isdeleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:10, 15, and 12. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:10 with three or less (e.g., three or less, two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, (b) the sequence set forth in SEQ ID NO:15 withfive or less (e.g., five or less, four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, and (c) the sequence set forth in SEQ ID NO:12with two or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:17, with the exception that the first threonine residue or the lasttyrosine residue of SEQ ID NO:17 is deleted or replaced with a differentamino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:14, 11, and 12. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:14 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:11 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:12 with two or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:18, with the exception thatthe first serine residue or the last tyrosine residue of SEQ ID NO:18 isdeleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:14, 11, and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:14 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:11 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:19 with two or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:20, with the exception thatthe first serine residue or the last histidine residue of SEQ ID NO:20is deleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:21, 11, and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:21 with three or less (e.g., three or less, two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, (b) the sequence set forth in SEQ ID NO:11 withfive or less (e.g., five or less, four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, and (c) the sequence set forth in SEQ ID NO:19with two or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:22, with the exception that the first serine residue or the lasthistidine residue of SEQ ID NO:22 is deleted or replaced with adifferent amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:23, 11, and 7. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:23 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:11 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:7 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof. Forexample, a polypeptide provided herein can contain the sequence setforth in SEQ ID NO:24, with the exception that the first glutamic acidresidue or the last alanine residue of SEQ ID NO:24 is deleted orreplaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:10, 11, and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:10 with three or less (e.g., three or less, two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, (b) the sequence set forth in SEQ ID NO:11 withfive or less (e.g., five or less, four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, and (c) the sequence set forth in SEQ ID NO:19with two or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:25, with the exception that the first threonine residue or the lasthistidine residue of SEQ ID NO:25 is deleted or replaced with adifferent amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:26, 3, and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:26 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:3 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:19 with two or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:27, with the exception thatthe first arginine residue or the last histidine residue of SEQ ID NO:27is deleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:26, 28, and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:26 with two or less (e.g., two or less, one, or zero) aminoacid additions, deletions, substitutions, or combinations thereof, (b)the sequence set forth in SEQ ID NO:28 with five or less (e.g., five orless, four or less, three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, and (c)the sequence set forth in SEQ ID NO:19 with two or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:29, with the exception thatthe first arginine residue or the last histidine residue of SEQ ID NO:29is deleted or replaced with a different amino acid residue.

In some cases, a polypeptide can include the sequences set forth in SEQID NOS:28 and 19. In some cases, a polypeptide provided herein cancontain an amino acid sequence that aligns to (a) the sequence set forthin SEQ ID NO:28 with five or less (e.g., five or less, four or less,three or less, two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof, and (b) the sequenceset forth in SEQ ID NO:19 with two or less (e.g., two or less, one, orzero) amino acid additions, deletions, substitutions, or combinationsthereof. For example, a polypeptide provided herein can contain thesequence set forth in SEQ ID NO:30, with the exception that the firstglycine residue or the last histidine residue of SEQ ID NO:30 is deletedor replaced with a different amino acid residue.

A polypeptide provided herein can have any length. For example, apolypeptide provided herein can be between 17 and 45 (e.g., between 18and 40, between 22 and 44, between 25 and 45, between 26 and 44, between27 and 43, between 28 and 42, between 29 and 41, between 30 and 40,between 31 and 39, between 23 and 35, between 25 and 30, or between 30and 35) amino acid residues in length. It will be appreciated that apolypeptide with a length of 17 or 45 amino acid residues is apolypeptide with a length between 17 and 45 amino acid residues.

Variant NPs having conservative and/or non-conservative substitutions(e.g., with respect to any of SEQ ID NOS:1 to 30), as well as fragmentsof any of SEQ ID NOS:1 to 30, fragments of variants of any of SEQ IDNOS:1 to 30, and polypeptides comprising any of SEQ ID NOS:1 to 30,variants or fragments of any of SEQ ID NOS:1 to 30, or fragments ofvariants of any of SEQ ID NOS:1 to 30, can be screened for biologicalactivity using any suitable assays, including those described herein.For example, the activity of a NP as described herein can be evaluatedin vitro by testing its ability to suppress proliferation of HAoSMC.Cells can be exposed to a NP (e.g., 10⁻⁹ to 10⁻⁴ M ANP, BNP, CNP, CD-NP,CU-NP, ABC-NP, ABC-NP1, or BC-NP2), and samples can be assayed toevaluate the NP effects on cell proliferation. Cell proliferation can bedetected and measured using, for example, a colormetricbromodeoxyuridine (BrdU) cell proliferation ELISA (Roche, Indianapolis,Ind.).

The activity of a NP also can be evaluated in vivo by, for example,testing its effects on factors such as pulmonary capillary wedgepressure, right atrial pressure, mean arterial pressure, urinary sodiumexcretion, urine flow, proximal and distal fractional sodiumreabsorption, plasma renin activity, plasma cGMP levels, urinary cGMPexcretion, net renal generation of cGMP, glomerular filtration rate, andleft ventricular mass in animals. In some cases, such parameters can beevaluated after induced MI (e.g., MI induced by coronary arteryligation).

In some embodiments, the NPs provided herein can be cyclic due todisulfide bonds between cysteine residues (see, e.g., the ANP, BNP, CNP,and DNP structures depicted in FIG. 1). In some embodiments, asulfhydryl group on a cysteine residue can be replaced with analternative group (e.g., —CH₂CH₂—). To replace a sulfhydryl group with a—CH₂-group, for example, a cysteine residue can be replaced byalpha-aminobutyric acid. Such cyclic analog polypeptides can begenerated, for example, in accordance with the methodology of Lebl andHruby ((1984) Tetrahedron Lett. 25:2067), or by employing the proceduredisclosed in U.S. Pat. No. 4,161,521.

In addition, ester or amide bridges can be formed by reacting the OH ofserine or threonine with the carboxyl group of aspartic acid or glutamicacid to yield a bridge having the structure —CH₂CO₂CH₂—. Similarly, anamide can be obtained by reacting the side chain of lysine with asparticacid or glutamic acid to yield a bridge having the structure—CH₂C(O)NH(CH)₄—. Methods for synthesis of these bridges are known inthe art (see, e.g., Schiller et al. (1985) Biochem. Biophy. Res. Comm.127:558, and Schiller et al. (1985) Int. J. Peptide Protein Res.25:171). Other bridge-forming amino acid residues and reactions areprovided in, for example, U.S. Pat. No. 4,935,492. Preparation ofpeptide analogs that include non-peptidyl bonds to link amino acidresidues also are known in the art. See, e.g., Spatola et al. (1986)Life Sci. 38:1243; Spatola (1983) Vega Data 1(3); Morley (1980) TrendsPharm. Sci. 463-468; Hudson et al. (1979) Int. J. Pept. Prot. Res.14:177; Spatola, in Chemistry and Biochemistry of Amino Acid Peptidesand Proteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983);Hann (1982) J. Chem. Soc. Perkin Trans. 1:307; Almquist et al. (1980) J.Med. Chem. 23:1392; Jennings-White et al. (1982) Tetrahedron Lett.23:2533; European Patent Application EP 45665; Holladay et al. (1983)Tetrahedron Lett. 24:4401; and Hruby (1982) Life Sci. 31:189.

In some embodiments, a NP can comprise an amino acid sequence as setforth in SEQ ID NOS:1, 2, 3, 4, 5, 8, 9, 13, 16, 17, 18, 20, 22, 24, 25,27, 29, or 30, but with a particular number of amino acid substitutions.For example, a NP can have the amino acid sequence of any one of SEQ IDNOS:1, 2, 3, 4, 5, 8, 9, 13, 16, 17, 18, 20, 22, 24, 25, 27, 29, or 30,but with one, two, three, four, or five amino acid substitutions.Examples of such amino acid sequences include, without limitation, thoseset forth in SEQ ID NOS:31-150.

In some embodiments, a NP as provided herein can include an amino acidsequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99.0%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, or 100%) sequence identity to a region of areference NP sequence (e.g., SEQ ID NOS:1-30). Percent sequence identityis calculated by determining the number of matched positions in alignedamino acid sequences, dividing the number of matched positions by thetotal number of aligned amino acids, and multiplying by 100. A matchedposition refers to a position in which identical amino acids occur atthe same position in aligned amino acid sequences. Percent sequenceidentity also can be determined for any nucleic acid sequence.

Percent sequence identity is determined by comparing a target nucleicacid or amino acid sequence to the identified nucleic acid or amino acidsequence using the BLAST 2 Sequences (Bl2seq) program from thestand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained on the World Wide Web from Fish & Richardson's web site(fr.com/blast) or the U.S. government's National Center forBiotechnology Information web site (ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt-j c:\seq2.txt -p blastn -o c:\output.txt -q −1-r 2. If the targetsequence shares homology with any portion of the identified sequence,then the designated output file will present those regions of homologyas aligned sequences. If the target sequence does not share homologywith any portion of the identified sequence, then the designated outputfile will not present aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (1) a target sequence that is 30 amino acids in lengthis compared to the sequence set forth in SEQ ID NO:2, (2) the Bl2seqprogram presents 27 amino acids from the target sequence aligned with aregion of the sequence set forth in SEQ ID NO:2 where the first and lastamino acids of that 27 amino acid region are matches, and (3) the numberof matches over those 27 aligned amino acids is 25, then the 30 aminoacid target sequence contains a length of 27 and a percent identity overthat length of 92.6 (i.e., 25) 27×100=92.6).

It will be appreciated that different regions within a single amino acidor nucleic acid target sequence that aligns with an identified sequencecan each have their own percent identity. It is noted that the percentidentity value is rounded to the nearest tenth. For example, 78.11,78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16,78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted thatthe length value will always be an integer.

Isolated polypeptides can be produced using any suitable methods,including solid phase synthesis, and can be generated using manualtechniques or automated techniques (e.g., using an Applied BioSystems(Foster City, Calif.) Peptide Synthesizer or a Biosearch Inc. (SanRafael, Calif.) automatic peptide synthesizer. Disulfide bonds betweencysteine residues can be introduced by mild oxidation of the linearpolypeptides using KCN as taught, e.g., in U.S. Pat. No. 4,757,048. NPsalso can be produced recombinantly, as described herein.

In some cases, a polypeptide provided herein can be a substantially purepolypeptide. As used herein, the term “substantially pure” withreference to a polypeptide means that the polypeptide is substantiallyfree of other polypeptides, lipids, carbohydrates, and nucleic acid withwhich it is naturally associated. Thus, a substantially pure polypeptideis any polypeptide that is removed from its natural environment and isat least 60 percent pure or is any chemically synthesized polypeptide. Asubstantially pure polypeptide can be at least about 60, 65, 70, 75, 80,85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

Salts of carboxyl groups of polypeptides can be prepared by contactingthe peptide with one or more equivalents of a desired base such as, forexample, a metallic hydroxide base (e.g., sodium hydroxide), a metalcarbonate or bicarbonate base (e.g., sodium carbonate or sodiumbicarbonate), or an amine base (e.g., triethylamine, triethanolamine,and the like). Acid addition salts of polypeptides can be prepared bycontacting the polypeptide with one or more equivalents of an inorganicor organic acid (e.g., hydrochloric acid).

Esters of carboxyl groups of polypeptides can be prepared using anysuitable means (e.g., those known in the art) for converting acarboxylic acid or precursor to an ester. For example, one method forpreparing esters of the present polypeptides, when using the Merrifieldsynthesis technique, is to cleave the completed polypeptide from theresin in the presence of the desired alcohol under either basic oracidic conditions, depending upon the resin. The C-terminal end of thepolypeptide then can be directly esterified when freed from the resin,without isolation of the free acid.

Amides of polypeptides can be prepared using techniques (e.g., thoseknown in the art) for converting a carboxylic acid group or precursor toan amide. One method for amide formation at the C-terminal carboxylgroup includes cleaving the polypeptide from a solid support with anappropriate amine, or cleaving in the presence of an alcohol, yieldingan ester, followed by aminolysis with the desired amine.

N-acyl derivatives of an amino group of a polypeptide can be prepared byutilizing an N-acyl protected amino acid for the final condensation, orby acylating a protected or unprotected peptide. O-acyl derivatives canbe prepared for example, by acylation of a free hydroxy peptide orpeptide resin. Either acylation may be carried out using standardacylating reagent such as acyl halides, anhydrides, acyl imidazoles, andthe like. Both N- and O-acylation may be carried out together, ifdesired.

In some embodiments, the NPs provided herein can have half-lives thatare increased relative to the half-life of native NPs. For example,while the half-life of CNP after administration to a mammal is short(about a minute and a half), the elimination half-life of a chimeric NPsuch as CUB-NP or CAB-NP can be increased. ANP provided herein can havea half life that is increased by at least 2-fold (e.g., at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold) ascompared to a native NP such as CNP, for example. In some embodiments, aNP can have an elimination half-life of at least about 10 minutes (e.g.,at least about 10 minutes, at least about 12 minutes, at least about 15minutes, at least about 17 minutes, at least about 18 minutes, or atleast about 20 minutes).

In some embodiments, a NP can be modified by linkage to a polymer suchas polyethylene glycol (PEG), or by fusion to another polypeptide suchas albumin, for example. For example, one or more PEG moieties can beconjugated to a NP via lysine residues. Linkage to PEG or anothersuitable polymer, or fusion to albumin or another suitable polypeptidecan result in a modified NP having an increased half life as compared toan unmodified NP. Without being bound by a particular mechanism, anincreased serum half life can result from reduced proteolyticdegradation, immune recognition, or cell scavanging of the modified NP.Methods for modifying a polypeptide by linkage to PEG (also referred toas “PEGylation”) or other polymers are known in the art, and includethose set forth in U.S. Pat. No. 6,884,780; Cataliotti et al. ((2007)Trends Cardiovasc. Med. 17:10-14; Veronese and Mero (2008) BioDrugs22:315-329; Miller et al. (2006) Bioconjugate Chem. 17:267-274; andVeronese and Pasut (2005) Drug Discov. Today 10:1451-1458, all of whichare incorporated herein by reference in their entirety. Methods formodifying a polypeptide by fusion to albumin also are known in the art,and include those set forth in U.S. Patent Publication No. 20040086976,and Wang et al. (2004) Pharm. Res. 21:2105-2111, both of which areincorporated herein by reference in their entirety.

A NP as provided herein can function through the one or more of theguanylyl cyclase receptors through which the native NPs function. Forexample, in some embodiments, a NP as provided herein can bind to andfunction through the NPR-A receptor through which ANP and BNP function.In some cases, a NP can bind to and function through the NPR-A receptor,as do ANP and BNP. In some cases, a NP as provided herein can functionthrough the NPR-B receptor through which CNP functions. In some cases, aNP as provided herein can bind to and function through the NPR-Creceptor. Further, in some cases, a NP as provided herein can bind toand function through more than one guanylyl cyclase receptor, includingNPR-A and NPR-B, for example. Methods for evaluating which receptor isinvolved in function of a particular NP are known in the art. Forexample, glomeruli, which contain both NPR-A and NPR-B, can be isolated(e.g., from a laboratory animal such as a dog) and incubated with a NP(e.g., a native, chimeric, or mutated NP), and cGMP levels can bemeasured. Glomeruli can be pretreated with antagonists of NPR-A or NPR-Bto determine whether cGMP production stimulated by a NP through one orthe other receptor can be attenuated.

In some cases, compounds (e.g., isolated NPs) provided herein can reduceor prevent restenosis. To determine whether a particular compound hasthe ability to reduce or prevent restenosis, one can carry out assaysthat are well known in the art, including those described herein (e.g.,in the Example below).

Nucleic Acids, Vectors, and Host Cells

This document also describes exemplary nucleic acids encodingpolypeptides (e.g., NPs), as well as expression vectors containing thenucleic acids, and host cells containing the nucleic acids and/orexpression vectors. As used herein, the term “nucleic acid” refers toboth RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. A nucleic acid molecule can bedouble-stranded or single-stranded (i.e., a sense or an antisense singlestrand). Nucleic acids include, for example, cDNAs encoding the NPs,variant NPs, and chimeric NPs provided herein.

An “isolated nucleic acid” is a nucleic acid that is separated fromother nucleic acid molecules that are present in a vertebrate genome,including nucleic acids that normally flank one or both sides of thenucleic acid in a vertebrate genome. The term “isolated” as used hereinwith respect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid. By wayof example and not limitation, an “isolated ANP nucleic acid,” forexample, can be a RNA or DNA molecule containing 9 or more (e.g., 15 ormore, 21 or more, 36 or more, or 45 or more) sequential nucleotide basesthat encode at least a portion of ANP, or a RNA or DNA complementarythereto.

Also provided herein are nucleic acid molecules that can selectivelyhybridize under stringent hybridization conditions to a nucleic acidmolecule encoding a NP (e.g., nucleic acid molecules encodingpolypeptides having the amino acid sequences set forth in SEQ ID NOS:1,2, 3, 4, 5, 8, 9, 13, 16, 17, 18, 20, 22, 24, 25, 27, 29, and 30, orvariants and fragments thereof. The term “selectively hybridize” meansto detectably and specifically bind under hybridization and washconditions that minimize appreciable amounts of detectable binding tononspecific nucleic acids. For example, high stringency conditions canbe used to achieve selective hybridization conditions. Moderate andstringent hybridization conditions include those that are well known inthe art. See, for example, sections 9.47-9.51 of Sambrook et al. (1989).As used herein, stringent conditions are those that (1) employ low ionicstrength and high temperature for washing, such as 0.015 M NaCl/0.0015 Msodium citrate (SSC) with 0.1% sodium lauryl sulfate (SDS) at 50° C., or(2) employ a denaturing agent such as formamide during hybridization,such as 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Alternatively, 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium phosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at42° C. can be used, with washes at 42° C. in 0.2×SSC and 0.1% SDS.

Isolated nucleic acid molecules can be produced using standardtechniques, including, without limitation, common molecular cloning andchemical nucleic acid synthesis techniques. For example, polymerasechain reaction (PCR) techniques can be used to obtain an isolatednucleic acid containing nucleotide sequence that encodes a NP asprovided herein. PCR refers to a procedure or technique in which targetnucleic acids are enzymatically amplified. Sequence information from theends of the region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers typically are 14 to 40nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. General PCR techniques are described, for examplein PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler,Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complementaryDNA (cDNA) strands. Ligase chain reaction, strand displacementamplification, self-sustained sequence replication, or nucleic acidsequence-based amplification also can be used to obtain isolated nucleicacids. See, for example, Lewis (1992) Genetic Engineering News 12:1;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; andWeiss (1991) Science 254:1292.

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids (e.g., nucleic acids encoding variant NPs) alsocan be obtained by mutagenesis. For example, a reference sequence can bemutated using standard techniques including oligonucleotide-directedmutagenesis and site-directed mutagenesis through PCR. See, ShortProtocols in Molecular Biology, Chapter 8, Green Publishing Associatesand John Wiley & Sons, edited by Ausubel et al., 1992. Non-limitingexamples of variant NPs art provided herein.

This document also contemplates nucleic acid molecules encoding aminoacid sequences from NPs other than ANP, BNP, CNP, DNP, URO, or chimerasor variants thereof. Sources of nucleotide sequences from which nucleicacid molecules encoding a NP, or the nucleic acid complement thereof,can be obtained include total or polyA+ RNA from any eukaryotic source,including reptilian (e.g., snake) or mammalian (e.g., human, rat, mouse,canine, bovine, equine, ovine, caprine, or feline) cellular source fromwhich cDNAs can be derived by methods known in the art. Other sources ofthe nucleic acid molecules provided herein include genomic librariesderived from any eukaryotic cellular source, including mammalian sourcesas exemplified above.

Nucleic acid molecules encoding native NPs can be identified andisolated using standard methods, e.g., as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY (1989). For example, reverse-transcriptase PCR(RT-PCR) can beused to isolate and clone NP cDNAs from isolated RNA that contains RNAsequences of interest (e.g., total RNA isolated from human tissue).Other approaches to identify, isolate and clone NP cDNAs include, forexample, screening cDNA libraries.

Vectors containing nucleic acids such as those described herein also areprovided. A “vector” is a replicon, such as a plasmid, phage, or cosmid,into which another DNA segment may be inserted so as to bring about thereplication of the inserted segment. An “expression vector” is a vectorthat includes one or more expression control sequences, and an“expression control sequence” is a DNA sequence that controls andregulates the transcription and/or translation of another DNA sequence.

In the expression vectors provided herein, a nucleic acid (e.g., anucleic acid encoding a NP) can be operably linked to one or moreexpression control sequences. As used herein, “operably linked” meansincorporated into a genetic construct so that expression controlsequences effectively control expression of a coding sequence ofinterest. Examples of expression control sequences include promoters,enhancers, and transcription terminating regions. A promoter is anexpression control sequence composed of a region of a DNA molecule,typically within 100 to 500 nucleotides upstream of the point at whichtranscription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence. Expression vectors thus can be useful to produceantibodies as well as other multivalent molecules.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

Host cells containing vectors also are provided. The term “host cell” isintended to include prokaryotic and eukaryotic cells into which arecombinant expression vector can be introduced. As used herein,“transformed” and “transfected” encompass the introduction of a nucleicacid molecule (e.g., a vector) into a cell by one of a number oftechniques. Although not limited to a particular technique, a number ofthese techniques are well established within the art. Suitable methodsfor transforming and transfecting host cells can be found, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual (2^(nd)edition), Cold Spring Harbor Laboratory, New York (1989). For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transfercan be used introduce nucleic acid into cells. In addition, naked DNAcan be delivered directly to cells in vivo as described elsewhere (U.S.Pat. Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document provides methods and materials for detecting a polypeptideprovided herein. Such methods and materials can be used to monitorpolypeptide levels within a mammal receiving the polypeptide as atherapeutic. A NP provided herein can be detected, for example,immunologically, using one or more antibodies. As used herein, the term“antibody” includes intact molecules as well as fragments thereof thatare capable of binding to an epitopic determinant of a polypeptideprovided herein. The term “epitope” refers to an antigenic determinanton an antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains, and typically havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Epitopes generally have at least fivecontiguous amino acids (a continuous epitope), or alternatively can be aset of noncontiguous amino acids that define a particular structure(e.g., a conformational epitope). The term “antibody” includespolyclonal antibodies, monoclonal antibodies, humanized or chimericantibodies, single chain Fv antibody fragments, Fab fragments, andF(ab)₂ fragments. Polyclonal antibodies are heterogeneous populations ofantibody molecules that are contained in the sera of the immunizedanimals. Monoclonal antibodies are homogeneous populations of antibodiesto a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a NP providedherein can be generated by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule;Fab fragments can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. In some cases, Fab expression libraries can beconstructed. See, for example, Huse et al., Science, 246:1275 (1989).Once produced, antibodies or fragments thereof can be tested forrecognition of a polypeptide provided herein by standard immunoassaymethods including ELISA techniques, radioimmunoassays, and Westernblotting. See, Short Protocols in Molecular Biology, Chapter 11, GreenPublishing Associates and John Wiley & Sons, Ed. Ausubel et al., 1992.

In immunological assays, an antibody having specific binding affinityfor a polypeptide provided herein or a secondary antibody that binds tosuch an antibody can be labeled, either directly or indirectly. Suitablelabels include, without limitation, radionuclides (e.g. ¹²⁵I, ¹³¹I, ³⁵S,³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, FITC,PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot™nanoparticles supplied by Invitrogen (Carlsbad, Calif.)), compounds thatabsorb light of a defined wavelength, or enzymes (e.g., alkalinephosphatase or horseradish peroxidase). Antibodies can be indirectlylabeled by conjugation with biotin then detected with avidin orstreptavidin labeled with a molecule described above. Methods ofdetecting or quantifying a label depend on the nature of the label andare known in the art. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting a polypeptide provided herein can beperformed in a variety of known formats, including sandwich assays,competition assays (competitive RIA), or bridge immunoassays. See, forexample, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074.Methods of detecting a polypeptide provided herein generally includecontacting a biological sample with an antibody that binds to apolypeptide provided herein and detecting binding of the polypeptide tothe antibody. For example, an antibody having specific binding affinityfor a polypeptide provided herein can be immobilized on a solidsubstrate by any of a variety of methods known in the art and thenexposed to the biological sample. Binding of the polypeptide to theantibody on the solid substrate can be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).In some cases, the antibody can be labeled and detected as describedabove. A standard curve using known quantities of a polypeptide providedherein can be generated to aid in the quantitation of the levels of thepolypeptide.

In some embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate can be used to detect the presence,absence, or level of a polypeptide provided herein. The solid substratecan be contacted with the biological sample such that any polypeptide ofinterest in the sample can bind to the immobilized antibody. Thepresence, absence, or level of the polypeptide bound to the antibody canbe determined using a “detection” antibody having specific bindingaffinity for the polypeptide. In some embodiments, a capture antibodycan be used that has binding affinity for ANP, BNP, CNP, DNP, and/orURO, or a chimeric or variant polypeptide as described herein, and adetection antibody can be used that has specific binding affinity for aparticular polypeptide provided herein (e.g., a polypeptide having theamino acid sequence set forth in any of SEQ ID NOS:1, 2, 3, 4, 5, 8, 9,13, 16, 17, 18, 20, 22, 24, 25, 27, or 30). It is understood that insandwich assays, the capture antibody should not bind to the sameepitope (or range of epitopes in the case of a polyclonal antibody) asthe detection antibody. Thus, if a monoclonal antibody is used as acapture antibody, the detection antibody can be another monoclonalantibody that binds to an epitope that is either physically separatedfrom or only partially overlaps with the epitope to which the capturemonoclonal antibody binds, or a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturemonoclonal antibody binds. If a polyclonal antibody is used as a captureantibody, the detection antibody can be either a monoclonal antibodythat binds to an epitope that is either physically separated from orpartially overlaps with any of the epitopes to which the capturepolyclonal antibody binds, or a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturepolyclonal antibody binds. Sandwich assays can be performed as sandwichELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Antibodies having specific binding affinity for a polypeptide providedherein can be produced through standard methods. For example, apolypeptide can be recombinantly produced as described above, can bepurified from a biological sample (e.g., a heterologous expressionsystem), or can be chemically synthesized, and used to immunize hostanimals, including rabbits, chickens, mice, guinea pigs, or rats. Forexample, a polypeptide having the amino acid sequence set forth in anyof SEQ ID NOS:1, 2, 3, 4, 5, 8, 9, 13, 16, 17, 18, 20, 22, 24, 25, 27,29, 30, or fragments or variants thereof that are at least six aminoacids in length, can be used to immunize an animal. Various adjuvantsthat can be used to increase the immunological response depend on thehost species and include Freund's adjuvant (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies canbe prepared using a polypeptide provided herein and standard hybridomatechnology. In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described by Kohler et al.,Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor etal., Immunology Today, 4:72 (1983); Cote et al., Proc. Natl. Acad. Sci.USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96 (1983)). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the monoclonal antibodies can be cultivated in vitroand in vivo.

Other techniques for detecting a polypeptide provided herein includemass-spectrophotometric techniques such as electrospray ionization(ESI), and matrix-assisted laser desorption-ionization (MALDI). See, forexample, Gevaert et al., Electrophoresis, 22:1645-51 (2001); Chaurand etal., J. Am. Soc. Mass Spectrom., 10:91-103 (1999). Mass spectrometersuseful for such applications are available from Applied Biosystems(Foster City, Calif.); Bruker Daltronics (Billerica, Mass.); andAmersham Pharmacia (Sunnyvale, Calif.).

Compositions and Methods for Administration

The compounds described herein (e.g., native NPs, as well as chimericand variant NPs), or nucleic acids encoding the polypeptides describedherein, can be incorporated into compositions for administration to asubject (e.g., a subject suffering from or at risk for restenosis).Thus, this document provides, for example, the use of compounds asdescribed herein in the manufacture of medicaments for treating (e.g.,reducing) restenosis. Methods for formulating and subsequentlyadministering therapeutic compositions are well known to those in theart. Dosages typically are dependent on the responsiveness of thesubject to the compound, with the course of treatment lasting fromseveral days to several months, or until a suitable response isachieved. Persons of ordinary skill in the art routinely determineoptimum dosages, dosing methodologies and repetition rates. Optimumdosages can vary depending on the relative potency of an antibody, andgenerally can be estimated based on the EC₅₀ found to be effective in invitro and/or in vivo animal models. Compositions containing thecompounds (e.g., NPs) and nucleic acids provided herein may be givenonce or more daily, weekly, monthly, or even less often, or can beadministered continuously for a period of time (e.g., hours, days, orweeks). For example, a NP or a composition containing a NP can beadministered to a MI patient at a dose of at least about 0.01 ng NP/kgto about 100 mg NP/kg of body mass at or about the time of reperfusion,or can be administered continuously as an infusion beginning at or aboutthe time of reperfusion and continuing for one to seven days (e.g., at adose of about 0.01 ng NP/kg/minute to about 0.5 μg NP/kg/minute).

The NPs and nucleic acids can be admixed, encapsulated, conjugated orotherwise associated with other molecules, molecular structures, ormixtures of compounds such as, for example, liposomes, receptor or celltargeted molecules, or oral, topical or other formulations for assistingin uptake, distribution and/or absorption.

In some embodiments, a composition can contain a NP as provided hereinin combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include, for example,pharmaceutically acceptable solvents, suspending agents, or any otherpharmacologically inert vehicles for delivering antibodies to a subject.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include,without limitation: water; saline solution; binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose or dextrose and other sugars, gelatin, or calcium sulfate);lubricants (e.g., starch, polyethylene glycol, or sodium acetate);disintegrates (e.g., starch or sodium starch glycolate); and wettingagents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing molecules described herein can beadministered by a number of methods, depending upon whether local orsystemic treatment is desired. Administration can be, for example,parenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous (i.v.)drip); oral; topical (e.g., transdermal, sublingual, ophthalmic, orintranasal); or pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols), or can occur by a combination of such methods.Administration can be rapid (e.g., by injection) or can occur over aperiod of time (e.g., by slow infusion or administration of slow releaseformulations).

Compositions and formulations for parenteral, intrathecal orintraventricular administration include sterile aqueous solutions (e.g.,sterile physiological saline), which also can contain buffers, diluentsand other suitable additives (e.g., penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers).

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Formulations for topical administration include, for example, sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions in liquid or solid oil bases.Such solutions also can contain buffers, diluents and other suitableadditives. Pharmaceutical compositions and formulations for topicaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be useful. In some embodiments, transdermaldelivery of NPs as provided herein can be particularly useful. Methodsand compositions for transdermal delivery include those described in theart (e.g., in Wermeling et al. (2008) Proc. Natl. Acad. Sci. USA105:2058-2063; Goebel and Neubert (2008) Skin Pharmacol. Physiol.21:3-9; Banga (2007) Pharm. Res. 24:1357-1359; Malik et al. (2007) CurrDrug Deliv. 4:141-151; and Prausnitz (2006) Nat. Biotechnol.24:416-417).

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulized aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsion formulations are particularlyuseful for oral delivery of therapeutic compositions due to their easeof formulation and efficacy of solubilization, absorption, andbioavailability. Liposomes can be particularly useful due to theirspecificity and the duration of action they offer from the standpoint ofdrug delivery.

Compositions provided herein can contain any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to a subject, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof forthe relevant compound (e.g., NP). Accordingly, for example, thisdocument describes pharmaceutically acceptable salts of NPs, prodrugsand pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. A prodrug is a therapeutic agent that is prepared in aninactive form and is converted to an active form (i.e., drug) within thebody or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. The term “pharmaceutically acceptablesalts” refers to physiologically and pharmaceutically acceptable saltsof the NPs useful in methods provided herein (i.e., salts that retainthe desired biological activity of the parent NPs without impartingundesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); salts formed with organic acids (e.g., acetic acid, citricacid, oxalic acid, palmitic acid, or fumaric acid); and salts formedwith elemental anions (e.g., bromine, iodine, or chlorine).

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositions, suchas dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents, and stabilizers. Furthermore, the composition can bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings, penetration enhancers, andaromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the other componentswithin the compositions.

In some cases, a polypeptide provided herein can be formulated as asustained release dosage form. For example, a NP can be formulated intoa controlled release formulation. In some cases, coatings, envelopes, orprotective matrices can be formulated to contain one or more of thepolypeptides provided herein. Such coatings, envelopes, and protectivematrices can be used to coat indwelling devices such as stents,catheters, and peritoneal dialysis tubing. In some cases, a polypeptideprovided herein can incorporated into a polymeric substances, liposomes,microemulsions, microparticles, nanoparticles, or waxes.

Pharmaceutical formulations as disclosed herein, which can be presentedconveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients (i.e., the antibodies) with the desired pharmaceuticalcarrier(s). Typically, the formulations can be prepared by uniformly andintimately bringing the active ingredients into association with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. Formulations can be sterilized ifdesired, provided that the method of sterilization does not interferewith the effectiveness of the molecules(s) contained in the formulation.

Methods for Reducing or Preventing Restenosis

This document also provides methods for using compounds (e.g., NPs) asdisclosed herein for reducing or preventing restenosis. Thus, thecompounds and nucleic acid molecules provided herein can be administeredto a mammal (e.g., a human or a non-human mammal) in order to reduce orprevent restenosis that can occur, for example, after angioplasty, stentplacement, vascular surgery, cardiac surgery, or interventionalradiology. The composition or NP can be administered at any suitabledose, depending on various factors including, without limitation, theagent chosen and the patient characteristics. Administration can belocal or systemic.

In some embodiments, a NP or a composition containing a NP can beadministered at a dose of at least about 0.01 ng NP/kg to about 100 mgNP/kg of body mass (e.g., about 10 ng NP/kg to about 50 mg NP/kg, about20 ng NP/kg to about 10 mg NP/kg, about 0.1 ng NP/kg to about 20 ngNP/kg, about 3 ng NP/kg to about 10 ng NP/kg, or about 50 ng NP/kg toabout 100 μg/kg) of body mass, although other dosages also may providebeneficial results. In some cases, a composition containing a NP can beadministered as a continuous intravenous infusion beginning at or aboutthe time of reperfusion (i.e., at the time an occluded artery isopened), and continuing for one to seven days (e.g., one, two, three,four, five, six, or seven days). Such a composition can be administeredat a dose of, for example, about 0.1 ng NP/kg/minute to about 500 ngNP/kg/minute (e.g., about 0.5 ng NP/kg/minute, about 1 ng NP/kg/minute,about 2 ng NP/kg/minute, about 3 ng NP/kg/minute, about 5 ngNP/kg/minute, about 7.5 ng NP/kg/minute, about 10 ng NP/kg/minute, about12.5 ng NP/kg/minute, about 15 ng NP/kg/minute, about 20 ngNP/kg/minute, about 25 ng NP/kg/minute, about 30 ng NP/kg/minute, about50 ng NP/kg/minute, about 100 ng NP/kg/minute, or about 300 ngNP/kg/minute). In some embodiments, a composition containing a NP can beadministered before reperfusion (e.g., about one hour prior toreperfusion), either as one or more individual doses or as a continuousinfusion beginning about one hour prior to reperfusion). For example, acomposition can be administered beginning about one hour, about 45minutes, about 30 minutes, or about 15 minutes prior to reperfusion. Insome cases, a composition containing a NP as provided herein can beadministered after reperfusion (e.g., within about ten hours ofreperfusion), and can be administered either as one or more individualdoses or as a continuous infusion beginning within about ten hours ofreperfusion. For example, a composition can be administered about onehour, about two hours, about three hours, about four hours, about fivehours, about six hours, about seven hours, about eight hours, about ninehours, or about ten hours after reperfusion.

In some embodiments, a NP or a composition containing a NP can beadministered via a first route (e.g., intravenously) for a first periodof time, and then can be administered via another route (e.g., topicallyor subcutaneously) for a second period of time. For example, acomposition containing a NP can be intravenously administered to amammal (e.g., a human) at a dose of about 0.1 ng NP/kg/minute to about300 ng NP/kg/minute (e.g., about 1 ng NP/kg/minute to about 15 ngNP/kg/minute, about 3 ng NP/kg/minute to about 10 ng NP/kg/minute, orabout 10 ng NP/kg/minute to about 30 ng NP/kg/minute) for one to sevendays (e.g., one, two, three, four, five, six, or seven days), andsubsequently can be subcutaneously administered to the mammal at a doseof about 10 ng NP/kg/day to about 100 ng NP/kg/day (e.g., about 10 ngNP/kg/day, about 20 ng NP/kg/day, about 25 ng NP/kg/day, about 30 ngNP/kg/day, about 50 ng NP/kg/day, or about 100 ng NP/kg/day) for five to30 days (e.g., seven, 10, 14, 18, 21, 24, or 27 days).

The methods provided herein can include administering to a mammal aneffective amount of a NP (e.g., a native, chimeric, or variant NP) or anucleic acid encoding a NP, or an effective amount of a compositioncontaining such a molecule. As used herein, the term “effective amount”is an amount of a molecule or composition that is sufficient to reducethe occurrence of restenosis in a mammalian recipient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 99%, or 100%). The presence or extent of restenosis can beevaluated using methods known in the art, including, for example,angiogram.

In some embodiments, for example, an “effective amount” of a NP asprovided herein can be an amount that reduces restenosis in a treatedmammal by at least 10% as compared to the level of restenosis in themammal prior to administration of the NP or without administration ofthe NP (e.g., the level of restenosis after a previous angioplasty orstent placement procedure for example), or as compared to the level ofrestenosis in a control, untreated mammal. The level of restenosis canbe assessed using, for example, the methods described herein.

The invention will be further described in the following example, whichdoes not limit the scope of the invention described in the claims.

Example NPs Prevent Restenosis of Vasculature and Muscular Conduits

The effects of natural and chimeric natriuretic peptides onproliferation of HAoSMC were assessed. HAoSMC were incubated withincreasing concentrations of natural natriuretic peptides (ANP, BNP, orCNP) or chimeric natriuretic peptides (CD-NP, CU-NP, ABC-NP, ABC-NP1, orBC-NP2). Cell proliferation studies were performed. Briefly, HAoSMC(passages 4 through 6; Clonetics Corporation Inc., San Diego, Calif.)were grown to confluence in culture media supplemented with requiredgrowth factors, and were passaged in 96 well plates and incubated withsupplemented smooth muscle cell media for 24 hours. The supplementedmedium was replaced with basal smooth muscle cell media for 24 hours torender the cells quiescent. The cells were subsequently incubated in thepresence or absence of serum-supplemented media (5%), and in thepresence of serum-supplemented growth media along with exogenous NPs(10⁻⁹ to 10⁻⁴ M ANP, BNP, CNP, CD-NP, CU-NP, ABC-NP, ABC-NP1, or BC-NP2)for 24 hours. Proliferation was assessed by measuring bromodeoxyuridine(BrdU) uptake over the next 24 hours, as previously described (Schirgeret al. (2000) J. Am. Coll. Cardiol. 35:796-801.)

Each of the NPs evaluated was found to have antiproliferative actions onHAoSMC (FIG. 2). Increasing concentrations of ANP reduced proliferationby about 10 percent (10⁻⁷ M ANP), about 18 percent (10⁻⁶ M ANP), andabout 30 percent (10⁻⁵ M ANP). Increasing concentrations of BNP reducedproliferation by about 10 percent (10⁻⁷ M BNP), about 20 percent (10⁻⁶ MBNP), and about 25 percent (10⁻⁵ M BNP). Increasing concentrations ofCNP reduced proliferation by about 20 percent (10⁻⁷ M and 10⁻⁶ M CNP)and about 30 percent (10⁻⁵ M CNP). Increasing concentrations of CD-NPhad the greatest effect, decreasing proliferation by about 22 percent(10⁻⁸ M CD-NP), about 30 percent (10⁻⁷ M CD-NP), about 35 percent (10⁻⁶M CD-NP), and about 40 percent (10⁻⁵ M CD-NP). Increasing concentrationsof CU-NP reduced proliferation by about 8 percent (10⁻⁸ M CU-NP), about10 percent (10⁻⁷M CU-NP), about 15 percent (10⁻⁶ M CU-NP), and about 20percent (10⁻⁵ M CU-NP). ABC-NP, ABC-NP1, and BC-NP2 reducedproliferation by about 5 percent (10⁻⁵ M and 10⁻⁶ M ABC-NP, and 10⁻⁷ MBP-NP2), about 10 percent (10⁻⁷ M and 10⁻⁸ M ABC-NP, 10⁻⁶ M and 10⁻⁷ MABC-NP1, and 10⁻⁵ M and 10⁻⁶ M BC-NP2), or about 15 percent (10⁻⁵ M and10⁻⁸ M ABC-NP1). Thus, natural and chimeric NPs may be useful to preventrestenosis of the vasculature and muscular conduits.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-27. (canceled)
 28. A method for reducing restenosis in a subjectidentified as being in need thereof, comprising administering to saidsubject a therapeutically effective amount of a composition comprising apharmaceutically acceptable carrier and a natriuretic polypeptide underconditions wherein restenosis in said subject is reduced, wherein saidnatriuretic polypeptide comprises the amino acid sequence set forth inSEQ ID NO:27, or the amino acid sequence set forth in SEQ ID NO:27 withone, two, three, four, or five amino acid substitutions relative to thesequence set forth in SEQ ID NO:27.
 29. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27 with five amino acid substitutions relative to thesequence set forth in SEQ ID NO:27.
 30. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27 with four amino acid substitutions relative to thesequence set forth in SEQ ID NO:27.
 31. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27 with three amino acid substitutions relative to thesequence set forth in SEQ ID NO:27.
 32. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27 with two amino acid substitutions relative to thesequence set forth in SEQ ID NO:27.
 33. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27 with one amino acid substitution relative to thesequence set forth in SEQ ID NO:27.
 34. The method of claim 28, whereinsaid natriuretic polypeptide comprises the amino acid sequence set forthin SEQ ID NO:27.